Characterization of iMKs using flow cytometry and light and electron microscopy. (A) A representative flow cytometry dot plot of unstimulated iMKs showing a high expression level of glycoprotein GPIbα. (B-C) Representative flow cytometry dot plots showing no PAC1 binding to unstimulated iMKs (B) and pronounced binding of PAC1 (expression of activated αIIbβ3) to thrombin-stimulated iMKs. (D) In the presence of 10 mM EDTA (negative control), thrombin-stimulated iMKs show no PAC1 binding. (E-F) Representative histograms derived from the flow cytometry plots B and C, respectively, showing a high level of PAR-1 expression in both unstimulated and thrombin-stimulated iMKs (red peaks) and a low level of PAR-1 in the contaminating non-iMK cells not expressing GPIbα (negative control, blue peaks). (G-H) Representative scanning electron microscopy (G) and confocal light microscopy (H) images of individual unstimulated iMKs. Scale bars, 12 μm. (I) Diameters of unstimulated iMKs assessed using the confocal microscopy images shown in H (50 cells analyzed; results are presented as the median and interquartile range (IQR) between the 25th and 75th percentiles as well as 5th and 95th percentiles).